Reduced Glycopeptide Susceptibility Research Articles

New Antimicrobial Resistance Strategies: An Adaptive Resistance Network Conferring Reduced Glycopeptide Susceptibility in VISA.

Background: Vancomycin-intermediate Staphylococcus aureus (VISA) emerges typically in the healthcare-associated methicillin-resistant S. aureus and more rarely in community-acquired S. aureus (CA-MRSA). VISA is a serious concern for public health due to its association with persistent infections, the failure of vancomycin treatment, and poor clinical outcomes. Currently, the burden of VISA is somewhat high, even though vancomycin is the mainstay treatment for severe MRSA infections. The molecular mechanisms of reduced glycopeptide susceptibility in S. aureus are constantly under investigation but have still not yet been fully characterized. Methods: Our goal was to investigate the reduced glycopeptide susceptibility mechanisms emerging in a VISA CA-MRSA versus its vancomycin-susceptible (VSSA) CA-MRSA parents in a hospitalized patient undergoing glycopeptide treatment. Comparative integrated omics, Illumina MiSeq whole-genome sequencing (WGS), RNA-Seq, and bioinformatics were performed. Results: Through a comparison of VISA CA-MRSA vs. its VSSA CA-MRSA parent, mutational and transcriptomic adaptations were found in a pool of genes involved, directly or indirectly, in the biosynthesis of the glycopeptide target conferring or supporting the VISA phenotype, and its cross-resistance with daptomycin. This pool included key genes responsible for the biosynthesis of the peptidoglycan precursors, i.e., D-Ala, the D-Ala-D-Ala dipeptide termini of the pentapeptide, and its incorporation in the nascent pentapeptide, as key targets of the glycopeptide resistance. Furthermore, accessory glycopeptide-target genes involved in the pathways corroborated the key adaptations, and thus, supported the acquisition of the VISA phenotype i.e., transporters, nucleotide metabolism genes, and transcriptional regulators. Finally, transcriptional changes were also found in computationally predicted cis-acting small antisense RNA triggering genes related both to the key or accessory adaptive pathways. Conclusion: Our investigation describes an adaptive resistance pathway acquired under antimicrobial therapy conferring reduced glycopeptide susceptibility in a VISA CA-MRSA due to a comprehensive network of mutational and transcriptional adaptations in genes involved in pathways responsible for the biosynthesis of glycopeptide's target or supporters of the key resistance path.

β-Lactams enhance daptomycin activity against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium in in vitro pharmacokinetic/pharmacodynamic models.

Enterococcus faecalis and Enterococcus faecium are frequently resistant to vancomycin and β-lactams. In enterococcal infections with reduced glycopeptide susceptibility, combination therapy is often administered. Our objective was to conduct pharmacokinetic/pharmacodynamic (PK/PD) models to evaluate β-lactam synergy with daptomycin (DAP) against resistant enterococci. One E. faecalis strain (R6981) and two E. faecium strains (R6370 and 8019) were evaluated. DAP MICs were obtained. All strains were evaluated for response to LL37, an antimicrobial peptide, in the presence and absence of ceftaroline (CPT), ertapenem (ERT), and ampicillin (AMP). After 96 h, in vitro models were run simulating 10 mg DAP/kg body weight/day, 600 mg CPT every 8 h (q8h), 2 g AMP q4h, and 1 g ERT q24h, both alone and in combination against all strains. DAP MICs were 2, 4, and 4 μg/ml for strains R6981, R6370, and 8019, respectively. PK/PD models demonstrated bactericidal activity with DAP-CPT, DAP-AMP, and DAP-ERT combinations against strain 8019 (P < 0.001 and log10 CFU/ml reduction of >2 compared to any single agent). Against strains R6981 and R6370, the DAP-AMP combination demonstrated enhancement against R6370 but not R6981, while the combinations of DAP-CPT and DAP-ERT were bactericidal, demonstrated enhancement, and were statistically superior to all other regimens at 96 h (P < 0.001) against both strains. CPT, ERT, and AMP similarly augmented LL37 killing against strain 8019. In strains R6981 and R6370, CPT and ERT aided LL37 more than AMP (P < 0.001). Compared to DAP alone, combination regimens provide better killing and prevent resistance. Clinical research involving DAP combinations is warranted.

Treatment of bacteraemia: meticillin-resistant Staphylococcus aureus (MRSA) to vancomycin-resistant S. aureus (VRSA)

Around the world, Staphylococcus aureus remains a dominant cause of bacteraemia. Whilst meticillin resistance remains the major phenotype of concern, various levels of reduced glycopeptide susceptibility are emerging with increasing frequency. The most common MRSA phenotypes now have raised vancomycin MICs within the susceptible range (MICs of 1–2mg/L). This phenomenon, known as MIC creep, is hotly contested and often denied. Key to detecting MIC creep may be to examine isolates fresh, as freezing can allow reversion to wild-type MIC, presumably by loss of mutations. Treatment failure is common with vancomycin and it is uncertain whether higher doses are beneficial. At the other extreme, when enough mutations have accumulated, full VISA status is achieved, although this can also be unstable on storage. Heteroresistant and VISA strains can be considered the inevitable end result of continued MIC creep and are even more likely to fail glycopeptide treatment. Currently full vancomycin resistance is uncommon, with only approximately 20 strains described and confirmed worldwide. Empirical treatment for patients with undefined Gram-positive sepsis can undoubtedly be improved by knowledge of MRSA status, so this is a potential advantage of hospital admission screening. If a patient is risk-assessed or screen-positive for MRSA, and infection is not serious, then vancomycin or teicoplanin is appropriate empirical therapy, providing loading doses are given to achieve therapeutic concentrations immediately (trough 15mg/L). For life-threatening infections, the glycopeptides are inadequate unless the isolate is likely to be fully susceptible (Etest<1.5mg/L). If not, daptomycin (8–10mg/L) can be used as monotherapy but the MIC should be measured as soon as possible.

Prevalence of isolates with reduced glycopeptide susceptibility in orthopedic device-related infections due to methicillin-resistant Staphylococcus aureus

We evaluated, by an improved susceptibility testing method, the prevalence and significance of low-level glycopeptide resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to a previously described, retrospective cohort of patients treated for orthopedic device-related infections (ODRI) at the Geneva University Hospital between 2000 and 2008. Fifty-seven individual or multiple isolates were retrieved from 41 ODRI patients for glycopeptide susceptibility and clonality studies, including 20 patients with prosthetic joint (PJ) and 21 with osteosynthesis (OS) MRSA infections. Low-level glycopeptide resistance was detected by elevated teicoplanin or/and vancomycin minimum inhibitory concentrations (MICs ≥ 4 mg/L), as determined by a previously validated combination of macrodilution and agar dilution assays of improved sensitivity. MRSA isolates with elevated teicoplanin MICs were detected in 20/41 (49 %) ODRI patients at the onset or during the course of glycopeptide therapy, namely, in 10 of 20 patients with PJ and 10 of 21 patients with OS infections. Only one isolate developed a concomitant increase in vancomycin MIC during therapy. 13/20 (65 %) glycopeptide-intermediate S. aureus (GISA)-infected patients, including 7/10 (70 %) with PJ and 6/10 (60 %) with OS, experienced treatment failure. In contrast, therapy failed in only 5/21 (24 %) ODRI patients with non-GISA isolates (p = 0.012), including 2/10 (20 %) with PJ and 3/11 (27 %) with OS infections. The emergence of low-level teicoplanin resistance could not be explained by teicoplanin administration, since only four patients received teicoplanin. The evaluation of low-level teicoplanin resistance may improve the detection of GISA isolates. Further studies are warranted to evaluate the impact of low-level teicoplanin resistance on the outcome of glycopeptide therapy.

High Prevalence of Isolates with Reduced Glycopeptide Susceptibility in Persistent or Recurrent Bloodstream Infections Due to Methicillin-Resistant Staphylococcus aureus

Reduced susceptibility to glycopeptides in methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates is considered a risk factor for failure of glycopeptide therapy. We compared the prevalences of MRSA isolates with reduced glycopeptide susceptibility in patients with versus without persistent or recurrent MRSA bloodstream infections. A retrospective cohort study at the University Hospital of Geneva identified 27 patients with persistent or recurrent clonally related MRSA bacteremic episodes over an 8-year period, which included 208 consecutive nosocomial MRSA bacteremic episodes. Vancomycin and teicoplanin MICs were determined by a modified macrodilution assay allowing improved detection of glycopeptide-intermediate MRSA isolates (GISA), characterized by elevated teicoplanin or/and vancomycin MICs (≥ 4 μg/ml). For 16 patients (59%), their pretherapy and/or posttherapy MRSA isolates showed elevated teicoplanin MICs, among which 10 (37%) concomitantly displayed elevated vancomycin MICs. In contrast, 11 other patients (41%) were persistently or recurrently infected with non-GISA isolates. In comparison, only 39 (22%) of 181 single isolates from patients with no microbiological evidence of persistent or recurrent infections showed elevated teicoplanin MICs, among which 14 (8%) concomitantly displayed elevated vancomycin MICs. Clinical, microbiological, and pharmacokinetic variables for patients persistently or recurrently infected with GISA or non-GISA isolates were similar. Bacteremic patients with a poor response to glycopeptide therapy had a 2.8-fold- and 4.8-fold-higher rates of MRSA isolates displaying elevated teicoplanin and vancomycin MICs, respectively, than patients with single isolates (P < 0.0001). Detection of elevated teicoplanin MICs may help to predict a poor response to glycopeptide therapy in MRSA bacteremic patients.

Emergence of daptomycin resistance following vancomycin-unresponsive Staphylococcus aureus bacteraemia in a daptomycin-naïve patient—a review of the literature

A patient developed a daptomycin-resistant methicillin-resistant Staphylococcus aureus (MRSA) infection, despite being daptomycin-naïve, in the setting of persistent bacteraemia secondary to vertebral osteomyelitis. Modified population analysis profiling of sequential MRSA blood culture isolates revealed transition from a vancomycin-susceptible phenotype to a vancomycin-intermediate S. aureus (VISA) phenotype through a vancomycin-heteroresistant S. aureus (hVISA) intermediary. Increased cell wall thickening, determined by transmission electron microscopy, correlated with the emergence of daptomycin resistance. This case supports the current hypothesis that MRSA with reduced glycopeptide susceptibility are less susceptible to daptomycin because of a thickened cell wall. This may have significance for the use of daptomycin in salvage therapy. Other predictors of daptomycin resistance include bacteraemic persistence and the presence of high inoculum infections. As resistance may appear de novo and be unstable in vivo, all isolates should have daptomycin susceptibility testing performed. The optimal antibiotic option for salvage therapy of these daptomycin-resistant infections is unknown. However, these findings emphasise the importance of optimising management, including the consideration of early surgical intervention to avoid the emergence of daptomycin resistance, especially in high inoculum infections.

Staphylococcus aureus with reduced glycopeptide susceptibility in Liverpool, UK

To investigate if colonization with heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) is associated with hGISA bacteraemia. Isolates of methicillin-resistant S. aureus (MRSA) from blood cultures and from swabs to detect MRSA colonization were screened for reduced susceptibility to glycopeptides by an agar incorporation method. Isolates detected by this screen were tested for glycopeptide resistance by MacroEtest, standard MIC Etest methods and population analysis profile-AUC (PAP-AUC) analysis. S. aureus isolates with and without reduced glycopeptide susceptibility were characterized by PFGE and spa typing. MRSA isolates with reduced susceptibility to glycopeptides, as identified by the MacroEtest method, were detected in the colonization screens of 86 of 2550 MRSA-positive patients. The isolates were confirmed by Etest MIC and PAP-AUC analysis as hGISA. A total of 82/86 of the hGISA colonizing isolates were EMRSA-16 by PFGE; the remainder were EMRSA-15. Bacteraemia with hGISA was identified in five patients during the study period; two isolates were EMRSA-16 and three were EMRSA-15. hGISA colonization could not be linked to hGISA bacteraemia and hGISA bacteraemia could not be linked to hGISA colonization. Four of the five hGISA bacteraemias developed following teicoplanin therapy for a central venous catheter-associated MRSA bacteraemia. Laboratory strategies to reduce morbidity associated with hGISA should focus on testing for hGISA in bacteraemic (rather than colonizing) MRSA isolates in patients with recurrent S. aureus bacteraemia following glycopeptide exposure.

Selection of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus by Imipenem

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain Delta IP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS(H14)) that caused an amino acid substitution (S(329)-->L). This mutation is different from the VraS mutation (N(5)-->I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of DeltaIP with the mutated vraS(H14) gene confirmed that the S(329)-->L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N(5)-->I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of Delta IP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.

In vivo development of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA), GISA and daptomycin resistance in a patient with meticillin-resistant S. aureus endocarditis

We report a patient who developed a meticillin-resistant Staphylococcus aureus (MRSA) central venous catheter infection complicated by infective endocarditis. The patient was initially treated with glycopeptides, which led to the development of heterogeneous glycopeptide resistance, the detection of which required the use of a macro Etest screening test. Subsequently, the causative strain, confirmed by PFGE as a UK epidemic MRSA-15, was treated with daptomycin, and again resistance developed in vivo. The development in vivo of resistance to both these agents suggests that the resistance mechanisms may be associated. We suggest that the clinician managing MRSA infection should anticipate daptomycin resistance when reduced glycopeptide susceptibility is detected.

Teicoplanin pharmacodynamics in reference to the accessory gene regulator (agr) in Staphylococcus aureus using an in vitro pharmacodynamic model

The accessory gene regulator (agr) has been identified as playing a role in the expression of reduced glycopeptide susceptibility in Staphylococcus aureus. Previous studies indicate that strains with agr dysfunctional group II polymorphism have a higher propensity for reduced vancomycin activity. We investigated this relationship in agr groups I-IV using another glycopeptide, teicoplanin, in an in vitro pharmacodynamic model. Teicoplanin doses ranging from 1.88 to 30 mg/kg daily (fAUC/MIC 26.1-380.7) were simulated and evaluated for activity and the development of reduced susceptibility over 72 h. A dose-response relationship in activity was noted as doses escalated up to 30 mg/kg daily, but regrowth was identified with all doses. Teicoplanin doses of 3.75 and 1.88 mg/kg daily resulted in isolates with intermediate teicoplanin susceptibility (MIC = 16 mg/L) in agr groups II, IV (MIC = 16 mg/L) and III (MIC = 24 mg/L), regardless of function of the agr operon. Resistance to teicoplanin (> or = 32 mg/L) occurred in agr group I functional and dysfunctional isolates. Minimal changes in MIC were noted with 7.5 mg/kg daily doses in agr groups II-IV. However, this dose resulted in variable susceptibility (4-24 mg/L) in agr group I(+/-) isolates. Higher doses of 15 and 30 mg/kg daily did not produce changes in MIC in any isolate tested. Agr function did not determine teicoplanin resistance proclivity and is consistent with the previously described higher mutation rate in S. aureus to teicoplanin. Further investigation of agr group and function is warranted for all glycopeptides and compounds with a similar mechanism of action.

Proficiency of Italian clinical laboratories in detecting reduced glycopeptide susceptibility in Enterococcus and Staphylococcus spp. using routine laboratory methodologies

Objective To assess the ability of 59 clinical microbiology laboratories distributed throughout Italy to correctly identify and detect reduced susceptibility to glycopeptides in staphylococci and VanA-, VanB- or VanC-mediated glycopeptide resistance in enterococci.Methods Eight test strains comprising three staphylococci (S. aureus ATCC 29212 and two vancomycin-intermediate S. haemolyticus [11105301, 10030683Y]) and five enterococci (E. faecalis ATCC 29212, E. faecalis ATCC 51299 VanB, E. faecium AIB40 VanA, E. faecalis V583 VanB and E. gallinarum AIB39 VanC1) were distributed to 59 Italian clinical microbiology laboratories. Each isolate was blind-coded, and laboratories were instructed to identify the strains and test isolates for susceptibility to teicoplanin and vancomycin using their standard methods. Results were assessed against consensus test results obtained by a reference laboratory. In addition, to complement data interpretation, laboratories were asked to provide retrospective routine test results from their respective hospitals.Results All 59 laboratories participating in the study completed the susceptibility testing and provided data for analysis. A total of 53 laboratories provided retrospective routine data. Overall, laboratories were able to identify isolates to the genus level successfully. E. gallinarum and S. haemolyticus posed problems for species identification, with only 40.6 and 71.2%, respectively, of results reported correctly; most incorrect results were reported as ‘other species’. For enterococcal test strains, VanA phenotypes were detected correctly by 96.6% of laboratories; VanB by 30.5% (E. faecalis ATCC 51299) and 88.1% (E. faecalis V583); and VanC1 by 67.8%. For staphylococcal test strains, 28.8% (S. haemolyticus 11105301) and 23.7% (S. haemolyticus 10030683Y) of the laboratories were able to detect reduced susceptibility to vancomycin. Errors in detecting vancomycin resistance in VanB and VanC1 enterococci were made with all methods, most noticeably by disk diffusion users. For staphylococci, most errors in reporting vancomycin-intermediate resistance occurred with disk diffusion and VITEK (software version 5.04) users. Overall, considerably fewer errors occurred with the detection of teicoplanin resistance, especially for staphylococci. For 1999, routine results show that 41/1749 (2.4%) of E. faecium, 220/11 180 (2.0%) of E. faecalis, 29/24 927 (0.12%) of S. aureus and 54/22 102 (0.24%) of coagulase-negative staphylococci were reported as resistant to vancomycin.Conclusion Italian laboratories are able to identify staphylococci and enterococci adequately, although all methodologies used have problems in identifying E. gallinarum and coagulase-negative staphylococci to the species level. While VanA phenotypes were efficiently detected, problems were experienced in detecting VanB and VanC phenotypes.The majority of laboratories were unable to detect reduced vancomycin susceptibility in staphylococci adequately, especially with disk diffusion and older VITEK systems. Teicoplanin appeared useful as a marker for detecting vancomycin resistance, particularly with disk diffusion. Should enterococcal VanB or staphylococcal glycopeptide-intermediate phenotypes become prevalent in Italy, it is likely that they would be under-detected. New systems under development, such as VITEK2, should improve this situation.

Emergence, mechanism, and clinical implications of reduced glycopeptide susceptibility in Staphylococcus aureus.

In order to understand the mechanism(s) of the resistance/reduced susceptibility of Staphylococcus aureus to glycopeptide antibiotics, the current data on the modes of action of glycopeptides were reviewed. In addition, the different test systems for detecting vancomycin resistance and the clinical relevance of resistant Staphylococcus aureus were analyzed. Finally, strategies to prevent the nosocomial spread of these bacteria are presented, as are new therapeutic options.

Outbreak of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in a Parisian hospital.

Epidemiological relationships were investigated between 40 methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased glycopeptide susceptibility isolated from November 1998 to March 1999 from 39 patients (17 infected and 22 colonized patients) in nine wards of the Broussais Hospital, Paris, France. Reduced glycopeptide susceptibility was readily detected on brain heart infusion (BHI) agar containing 6 microg of teicoplanin per ml and on gradient plates, but not by the standard disk diffusion method. The MICs of vancomycin and teicoplanin, determined on BHI agar, were 4 and 8 to 32 microg/ml, respectively (standard antibiotic dilution), and 4 to 8 and 8 to 32 microg/ml, respectively (E-test). All strains were resistant to macrolides, aminoglycosides, tetracycline, rifampin, sulfonamides, and pefloxacin, showed reduced susceptibility to fusidic acid and fosfomycin, and were susceptible to trimethoprim and chloramphenicol. Pulsed-field gel electrophoresis and lysotyping revealed that a multidrug-resistant MRSA clone with decreased susceptibility to glycopeptides has been discretely endemic since at least 1996 in our institution, where it was responsible for an outbreak in November and December 1998.

Molecular epidemiology of Staphylococcus epidermidis in a neonatal intensive care unit over a three-year period.

Coagulase-negative staphylococci, especially Staphylococcus epidermidis, are increasingly important nosocomial pathogens, particularly in critically ill neonates. A 3-year prospective surveillance of nosocomial infections in a neonatal intensive care unit (NICU) was performed by traditional epidemiologic methods as well as molecular typing of microorganisms. The aims of the study were (i) to quantify the impact of S. epidermidis on NICU-acquired infections, (ii) to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species, (iii) to evaluate the use of different methods for the epidemiologic typing of the isolates, and (iv) to characterize the occurrence and the spread of staphylococci with decreased glycopeptide susceptibility. Results confirmed that S. epidermidis is one of the leading causes of NICU-acquired infections and that the reduced glycopeptide susceptibility, if investigated by appropriate detection methods such as population analysis, is more common than is currently realized. Typing of isolates, which can be performed effectively through molecular techniques such as pulsed-field gel electrophoresis but not through antibiograms, showed that many of these infections are due to clonal dissemination and, thus, are potentially preventable by strict adherence to recommended infection control practices and the implementation of programs aimed toward the reduction of the unnecessary use of antibiotics. These strategies are also likely to have a significant impact on the frequency of the reduced susceptibility of staphylococci to glycopeptides, since this phenomenon appears to be determined either by more resistant clones transmitted from patient to patient or, to a lesser extent, by strains that become more resistant as a result of antibiotic pressure.

Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides.

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 microg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.